Myosin S1 Fragment (smooth), Chicken gizzard

On Arrival: 4°C
Briefly centrifuge to collect the product at the bottom of the tube. Reconstituting a tube of CS-MYS05 with 180 ul of Milli-Q water will generate a 1.4 mg/ml stock of gizzard S1 myosin in the following buffer: 8 mM PIPES pH 7.0, 8 mM MgCl2, 5% (w/v) sucrose and1% (w/v) dextran. The protein should not be exposed to repeated freeze-thaw cycles. The lyophilized protein is stable at 4C desiccated(< 10% humidity) for 1 year.

* Limited stock available. If stock is not available, Cytoskeleton will produce a new batch upon request. Minimum order will apply. Inquire for more information.

Measurement of F-actin activated myosin ATPase activity
Identification/characterization of proteins or small molecules that affect myosin ATPase activity
Identification/characterization of proteins or small molecules that affect myosin / F- actin interaction

The biological activity of chicken gizzard S1 myosin can be determined from its rate of F-actin activated ATP hydrolysis. The assay is constructed by first polymerizing actin to form F-actin. Myosin is then added in substoichiometric amounts and there action is initiated with ATP.

The following major steps are recognized:
Step 1. Assemble required chemicals. (30min). Only if screening.
Step 2. Prepare F-actin polymer stock. (2h).
Step 3. Prepare Motor Mix and plate reader. (15min).
Step 4. Pipette Motor Mix into wells and start reaction/platereader. (10min).
F-actin polymer stock
1. Resuspend ADMK or AD99 with 0.5 ml of Buffer to 2 mg/ml, Buffer is 5mM Pipes-KOH, 100 uM ATP, 500uM DTT.
2. Place at RT for 30 min to depolymerize the actin oligos that form during concentration/lyophilization.
3. Then add 2 mM MgCl2 and 2 mM EGTA and incubate at RT for 1 h to polymerize. (shelf life 1h at RT).
Myosin ATPase assay
1. Dilute S1 myosin to 0.5 mg/ml with ice cold PM12 Buffer containing 1mM DTT.
2. Mix the following to make 0.49 ml of actin/myosin mixture:
250 ?l of F-actin (2 mg/ml in 5mM Pipes pH 7.5, 2 mM MgCl2, 58 ?M CaCl2, 2 mM EGTA, 100 ?M ATP, 500 ?M DTT). Replaced with equal volume PM12 Buffer for no Factin control.
120 ?l of PM12 Buffer
100 ?l 5x MSEG
5 ?l of 100x PNP
10 ul of S1 myosin (0.5 mg/ml)
3. Add 5 ul of 50 mM ATP and mix.
4. Incubate at 37C for 3 min.
5. Using the pre-warmed half area 96-well plate, pipette the following:
6. Pipette 10 ?l of Milli-Q water into each well.
7. Pipette 90 ?l of actin/myosin mixture per well.
8. Start protocol, 41 readings, 30 seconds apart, 37C , OD 360nm.
9. Calculate Vmax and compare non-activated to F-actin activated samples.

Diagrammatic representation of the myosin protein and its subfragments. Myosin is a hexameric protein consisting of two heavy chains and two light chains. Myosin can be proteolytically cleaved into heavy meromyosin (HMM) and light meromyosin (LMM) by ?-chymotrypsin in the presence of magnesium. In the presence of EDTA, however, ?-chymotrypsin produces the soluble myosin S1 fragment (3).